Drinking water is a significant predictor of Blastocystis infection among rural Malaysian primary schoolchildren.

Abstract

Blastocystis infection has a worldwide distribution especially among the disadvantaged population and immunocompromised subjects. This study was carried out to determine the prevalence and the association of Blastocystis infection with the socio-economic characteristics among 300 primary schoolchildren, living in rural communities in Lipis and Raub districts of Pahang state, Malaysia. Stool samples were collected and examined for the presence of Blastocystis using direct smear microscopy after in vitro cultivation in Jones' medium. The overall prevalence of Blastocystis infection was found to be as high as 25.7%. The prevalence was significantly higher among children with gastrointestinal symptoms as compared to asymptomatic children (x2 =4.246; P=0.039). Univariate and multivariate analyses showed that absence of a piped water supply (OR=3.13; 95% CI=1.78, 5.46; P<0.001) and low levels of mothers' education (OR=3.41; 95% CI=1.62, 7.18; P<0.01) were the significant predictors of Blastocystis infection. In conclusion, Blastocystis is prevalent among rural children and the important factors that determine the infection were the sources of drinking water and mothers' educational level. Interventions with provision of clean water supply and health education especially to mothers are required.

Full text

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Drinking water is a significant predictor of Blastocystis
infection among rural Malaysian primary schoolchildren
AWATIF M. ABDULSALAM1, INIT ITHOI1*, HESHAM M. AL-MEKHLAFI1,3,
ABDULHAMID AHMED1, JOHARI SURIN1and JOON-WAH MAK2
1Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
2School of Postgraduate Studies and Research, International Medical University, Bukit Jalil, 57000 Kuala Lumpur,
Malaysia
3Department of Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, Sana’a, Yemen
(Received 3 December 2011; revised 20 January and 3 February 2012; accepted 4 February 2012)
SUMMARY
Blastocystis infection has a worldwide distribution especially among the disadvantaged population and immunocompro-
mised subjects. This study was carried out to determine the prevalence and the association of Blastocystis infection with the
socio-economic characteristics among 300 primary schoolchildren, living in rural communities in Lipis and Raub districts
of Pahang state, Malaysia. Stool samples were collected and examined for the presence of Blastocystis using direct smear
microscopy after in vitro cultivation in Jones’ medium. The overall prevalence of Blastocystis infection was found to be as
high as 25·7%. The prevalence was significantly higher among children with gastrointestinal symptoms as compared to
asymptomatic children (x2=4·246; P=0·039). Univariate and multivariate analyses showed that absence of a piped water
supply (OR=3·13; 95% CI=1·78, 5·46; P<0·001) and low levels of mothers’ education (OR=3·41; 95% CI=1·62, 7·18;
P<0·01) were the significant predictors of Blastocystis infection. In conclusion, Blastocystis is prevalent among rural
children and the important factors that determine the infection were the sources of drinking water and mothers’ educational
level. Interventions with provision of clean water supply and health education especially to mothers are required.
Key words: Blastocystis, predictors, drinking water, schoolchildren, Malaysia.
INTRODUCTION
Blastocystis, an intestinal protozoan, has a world-
wide distribution and is often the most commonly
reported human intestinal protozoan in children and
adultsindevelopingcountries(StenzelandBoreham,
1996; Tan, 2008). Infection with Blastocystis is
believed to be associated with gastrointestinal symp-
toms including acute or chronic diarrhoea, which is
sometimes self-limited, and tenesmus, pruritus,
anorexia, nausea, vomiting, fever and eosinophilia
(Leder et al. 2005; Sohail and Fischer, 2005). Several
forms of Blastocystis are observed in in vitro culture
(i.e. vacuolar, avacuolar, multivacuolar, granular,
amoeboid, and cyst), the vacuolar form being mostly
recognized through microscopy. The fecal-oral route
is considered the main mode of transmission of
Blastocystis in day-care centres, institutions and
among residents of small communities (Stenzel and
Boreham, 1996). Many reports have shown that poor
hygiene, consumption of contaminated food or water
and close contact with animals are significant risk
factors of Blastocystis infection (Nimir, 1993; Tan,
2004;Yoshikawaetal.2009;BaldurssonandKaranis,
2011).
In Malaysia, there is increasing concern about
Blastocystis infection as previous studies have shown
that Blastocystis is prevalent among urban and rural
people and in water of rivers from recreational areas
(Noor Azian et al. 2007; Ithoi et al. 2011). However,
data on the predictors of infection is largely lacking.
Therefore, this study was carried out to determine
the prevalence of Blastocystis and to investigate its
association with the socio-economic characteristics of
schoolchildren in a rural community in the Pahang
State, Malaysia.
MATERIALS AND METHODS
Study areas and subjects
This cross-sectional study was conducted between
April and August 2010 among rural schoolchildren
aged 6–13 years in Lipis (located about 230 Km
northeast of Kuala Lumpur) and Raub (located
about 190 Km northeast of Kuala Lumpur) districts
of Pahang state, Malaysia. In Lipis (longitude
102.03°E, latitude 4.11°N), 2 primary schools were
involved in this study; the National School of Gahai
(for Malay, with a total enrolment of 96 pupils) and
the National School of Kuala Koyan (for Aborigines,
* Corresponding author: Department of Parasitology,
Faculty of Medicine, University of Malaya, 50603 Kuala
Lumpur,Malaysia.Tel:
+603 79674754. E-mail: init@um.edu.my, init.ithoi@
gmail.com
+60379674767.Fax:
1
Parasitology, Page 1 of 7.
doi:10.1017/S0031182012000340
© Cambridge University Press 2012

Page 2

with a total enrolment of 160 pupils). Similarly, 2
primary schools in Raub (longitude 101.52°E,
latitude 3.48°N) were involved, these being the
National School of Lubuk Kulit (for Malay, with a
total enrolment of 100 pupils) and the National
School of Satak (for Aborigines with a total enrol-
ment of 363 pupils). Each school is located approxi-
mately at the centre of the villages.
The villages of Malay and the aboriginal popu-
lationareclosetoeachotherandalmostallhouseshad
an electricity supply. Houses of aboriginal people are
made of wood or bamboo in which environmental
sanitation, as well as personal hygiene, are poor. On
the other hand, the housing conditions of Malay
people are better; the traditional Malay houses are
timber houses raised on stilts with wooden or
bamboo walls. However, there are many houses
built of bricks and concrete in the Malay villages.
Asinother parts ofMalaysia,theclimateisequatorial
with hot-humid conditions and rainfall throughout
the year. The vegetation is the thick rain forest type
and there are few water streams in the area.
All schoolchildren attending these schools during
the survey visits were invited to participate in this
study. We observed a higher absenteeism rate among
aboriginal compared to Malay schoolchildren in
which more than one third of the schoolchildren
were absent. Although, a sample size of 247 children
was estimated to give the study at least 95% power at
5% level of significance, we invited all children in
these schools to participate (universal sampling).
Hence, atotal of 300 (70% of those present during the
visits 300/429) primary schoolchildren (150 males
and 150 females) aged 6–12 years have agreed to
participate in this study.
Questionnaire
A pre-testedquestionnairewasused for the collection
of demographic and socio-economic data, environ-
mental factors, and history of gastrointestinal symp-
toms (Al-Mekhlafi et al. 2008). The children and
their parents were interviewed by a well-trained
assistant, in their home settings, to fill in the
questionnaire.
Detection of Blastocystis
Freshfaecal samples werecollected intowide-mouth,
screw-capped containers and then transported to the
Laboratory, Department of Parasitology, University
of Malaya, Kuala Lumpur. Approximately 50 mg of
feces were inoculated into a 15-ml screw-capped tube
containing 5·0 ml of complete Jones’ medium (Ithoi,
1999). All inoculated tubes were tightly closed,
placed in a rack and incubated at 37 °C. The medium
in each of these tubes was replaced with the new
complete Jones’ medium every alternate day starting
from day 2 of cultivation. This was carried out by
discardingabout4·0 mlofthemediumatthetoplevel
(withoutdisturbing thepellet) andreplacedby 4·0ml
of new complete Jones’ medium. The presence of
Blastocystis was observed daily for 14 days of
cultivation, by placing 1 drop of cultured sediment
onto a glass slide, covered with a cover-slip and
viewed (X100 and X400 objectives) under light
microscopy (Olympus BX51). Various sizes of
Blastocystis cells (2–15 μm) were seen as vacuolar
(most common), granular (many especially in old
stool culture tubes), amoeboid (vacuolar or granular
with pseudopodia) and cyst forms. Some of these
morphologies are shown in Fig. 1. The number of
positive tubes for Blastocystis was then recorded.
Statistical analysis
Data analysis was performed using the Statistical
Package for Social Sciences for Windows SPSS
(version 13, September 2004). A proportion was
used to present the prevalence of infections and other
categorical variables. The Chi-squaretestwasused to
test for associations between independent variables
and
Blastocystis
infection.
regression model was performed to identify the
significant predictors of infection. P<0·05 was
considered statistically significant.
Amultiplelogistic
Ethical issues
Prior to the stool collection, the objectives of this
study were explained to the headmasters, teachers
and children, and informed verbal consent was
obtained. The protocol of this study was approved
by the Medical Ethics Committee of the University
of Malaya Medical Centre, Kuala Lumpur.
RESULTS
A total of 300 primary schoolchildren (150 males and
150 females, aged 6–12 years, median age=9 years,
Interquartile Range=9–11 years) participated in this
study. General characteristics of the subjects, includ-
ing the demographic and socio-economic profiles, are
shown in Table 1. In comparison, household income
and the percentage of fathers who have at least 6 years
of formal education are significantly higher among
Malay compared to aboriginal people. On the other
hand, the percentage of working aboriginal mothers
(mostly farmers or rubber tapper) was significantly
higher than in Malay people (P<0·05). However,
there was no significant difference in the percentage
of mothers with at least 6 years of formal education
between both populations. The majority of aborigi-
nal worked as farmers, and almost half of the Malay
were either government employees or have their own
business.
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Awatif M. Abdulsalam and others

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Prevalence and distribution of Blastocystis infection
The overall prevalence of Blastocystis was 25·7%
(77/300), it was more frequent in males than females
though this difference was not statistically significant
(x2=2·950;P=0·086).Similarly,therewasnosignifi-
cant difference in the rate of infection according to
age (χ2=0·031; P=0·860). Moreover, the prevalence
of Blastocystis was higher among the aboriginal
children than the Malay but the difference was also
not statistically significant (29·3% compared to
20·2%; x2=3·126; P=0·077).
the symptoms, the prevalence of Blastocystis was
significantly higher among the participants who had
gastrointestinal symptoms as compared to asympto-
matic participants (34·7% compared to 22·7%;
x2=4·246; P=0·039), with abdominal pain and
diarrhoea as the most common symptoms (Fig. 2).
Stool specimens were also screened for the presence
of other intestinal parasitic infections, and the
children were found to be infected with Trichuris
trichiura (47·0%), Ascaris lumbricoides (20·7%), hook-
worm (1·7%),
Giardia
Entamoeba histolytica/dispar (4·3%). Except giardia-
sis, there was no significant association between
these infections and the gastrointestinal symptoms
(P >0·05). Regarding co-infections, only 2·3% of the
children had both Blastocystis and G. duodenalis,
while 5·3% had Blastocystis with Ascaris and/or
Trichuris. However, about half (49·4%; 38/77) of
the Blastocystis infections were single infections, and
were also found to be associated significantly with the
gastrointestinal symptoms (x2=4·861; P=0·027).
With regards to
duodenalis
(15·3%) and
Predictors of Blastocystis infection
Variables that may be associated with the prevalence
of Blastocystis infection were analysed using uni-
variate and multivariate analyses, and the results are
presented in Table 2. The results of univariate
analysis showed that the absence of a piped water
supply (x2=14·844; P<0·001) and the low level of
mothers’
education(x2=9·097;
P<0·01)were
Table 1. General characteristics of schoolchildren
participating in this study (n=300)
Characteristics
Frequency
(%)
Age groups (years)
4 10 years
> 10 years
Gender
Female
Male
Ethnicity
Malay
Aborigines
Socio-economic status
Fathers’ education (at least 6 years)
Mothers’ education (at least 6 years)
Lowhousehold income (<RM* 500)
Working mothers
Large family (>7 members)
Supplied with piped water
Presence of animals at household
50·7
49·3
50·0
50·0
39·7
60·3
77·0
78·7
44·3
42·0
64·3
52·7
71·7
* RM, Malaysian ringgits (US$1.00=RM3.00).
Fig. 1. Various forms of Blastocystis cells (X400) from culture of stool samples.
3 Blastocystis infection among Aboriginal children

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significantly associated with the prevalence of infec-
tion. On the other hand, other variables including
age, sex, race, family size, family monthly income,
parental employment status and the presence of
animals at the household showed no significant
association with the prevalence of Blastocystis infec-
tion. Logistic regression analysis confirmed that the
absence of a piped water supply and the low level of
mothers’educationwerethesignificant predictors for
the occurrence of Blastocystis infection. School-
children who do not have a tap water facility were
more than 3 times as likely to be infected (OR=3·13;
95% CI=1·78, 5·46; P<0·001). Likewise, children of
mothers with a low level of education were more
likely to be infected with Blastocystis as compared
to those of mothers with at least 6 years of
formal education (OR=3·41; 95% CI=1·62, 7·18;
P<0·01).
DISCUSSION
In the present study, the infection rate of Blastocystis
was 25·6%, which is within the range of prevalence
rate of previous local reports. Several Malaysian
researchers noted that the infection rate of this
protozoan was between 1·1% and 4·4% in children
with diarrhoea (Sinniah and Rajeswari, 1994; Menon
et al. 1999). Recently, an epidemiological survey
showed a prevalence of 52·3% in Aborigines living in
Pos Senderut, Pahang (Noor Azian et al. 2007). Pos
Senderut is a remote aboriginal settlement located
deep in the jungle with no road access, electricity,
piped water and sanitation facilities and this could
explain the higher prevalence of Blastocystis and
other intestinal parasites. Comparing our findings
with studies from other countries showed that the
prevalence reported by the present study was similar
to those reported among children in Iran (28·2%) and
Colombia (22·4%) (Daryani et al. 2006; Boeke et al.
2010). However, it is higher than that among school-
aged children from Thailand and India (Yaicharoen
et al. 2006; Rayan et al. 2010). By contrast,
considerably higher prevalence rates of 48·7% and
40·7%werereported among
Argentinean children, respectively (Baldo et al.
2004; Gamboa et al. 2011). Our findings showed no
significant difference in the prevalence of Blastocystis
infection according to age and gender of the
participants, and this is consistent with the results
of previous reports (Noor Azian et al. 2007;
Leelayoova et al. 2008; Cook et al. 2009).
Our findings also showed that the prevalence of
infection was higher among aboriginal schoolchil-
dren than their Malay peers and this could be
attributed to the poor living conditions and socio-
behavioural factors. The Aborigines were previously
living in isolated huts without electricity, toilet and
water supply, located near to the stream in the forest.
They were then re-settled along the water streams
and their houses were equipped with electricity and a
toilet. Some of the houses have a piped water supply.
Nevertheless, the Aborigines preferred defecating at
the site of the stream, and used water from streams
for most of their daily activities (swimming, cooking,
drinking, bathing and washing). This practice of
defecating near the streams
(especially children) in Lipis district, Pahang has
been noted by other workers (Al-Mekhlafi et al.
2008). Household animals such as dogs, cats and
chickens roamed freely in and out of the houses and
water streams, which could also contribute to the
contamination of water sources. The occurrence of
Blastocystis in water from wells and from the rain is
not known but Blastocystis, Cryptosporidium and
Philippiansand
by the residents
Fig. 2. Distribution of gastrointestinal symptoms among Blastocystis-infected schoolchildren.
4
Awatif M. Abdulsalam and others

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Giardia contaminated surface water from Malaysia
due to the exposure with infected feces from local
communities (human and animals) that live sur-
rounding the streams have been reported (Azman
et al. 2009; Ithoi et al. 2011). Blastocystis is
transmitted by the fecal-oral route among family
members, patients and staff of day-care centres, and
residents of small communities that lack access to
proper sanitation (Stenzel and Boreham, 1996).
As evidence of zoonotic transmission, several mol-
ecular studies have identified Blastocystis in a wide
range of animals and have reported that most of the
Blastocystis isolates from humans and animals in-
cluding monkeys, chickens, cattle, rats and pigs are
genetically identical (Yoshikawa et al. 2003, 2004,
2009).
As the first report from Malaysia, our study
indicates that children who have no access to a
piped water supply are more prone to get Blastocystis
infection, and this provides further evidence for
waterborne transmission of Blastocystis. Few studies
have implicated contaminated water as a source of
Blastocystis infections (Taamasri et al. 2000; Li et al.
2007; Leelayoova et al. 2008; Eroglu and Koltas,
2010; Baldursson and Karanis, 2011). This is not
surprising since the transmissible form ofthe parasite
is the water-resistant cyst (Tan, 2004). Blastocystis
cysts were reportedly able to survive in water for
up to 19 days at a normal temperature, but are fragile
at extremes of heat and cold, and in common
disinfectants (Zaman et al. 1995; Moe et al. 1996).
During the visits to the villages, we observed that
Table 2. Univariate and multivariate analyses of potential predictors associated with Blastocystis infection
among rural schoolchildren in Pahang, Malaysia (n=300)
(OR, odds ratio; CI, confidence interval.)
Variables
Blastocystis infection
Prevalence (%)OR (95% CI)
P
Age
410 years
>10 years
Gender
Male
Female
Ethnicity
Aborigines
Malay
Fathers’ education
No formal education
56 years formal education
Mothers’ education
No formal education
56 years formal education
Mothers’ employment status
Not working (housewives)
Working (public/private)
Household income
4RM*500/month (low)
>RM500/month
Family size
> 7 members (large)
47 members
Presence of toilet in house
No
Yes
Source of drinking water
Untreated (stream, rain, well)
Treated (piped)
Presence of animals at household
Yes
No
26·0
25·0
1·05 (0·60, 1·85)
1
0·860
30·0
21·3
1·58 (0·94, 2·67)
1
0·086
29·3
20·2
1·64 (0·95, 2·84)
1
0·077
31·9
23·8
1·50 (0·83, 2·70)
1
0·178
45·9
22·8
2·88 (1·15, 5·84)
1
0·003a,b
25·3
26·2
0·95 (0·57, 1·61)
1
0·860
29·3
22·8
1·41 (0·84, 2·37)
1
0·196
31·8
22·3
1·63 (0·98, 2·76)
1
0·071
26·0
24·7
1·07 (0·59, 1·93)
1
0·814
35·9
16·5
2·85 (1·65, 4·89)
1
<0·001a,b
24·7
28·2
0·83 (0·47, 1·46)
1
0·522
aSignificant association, P<0·05.
bConfirmed as significant predictors by logistic regression analysis.
* RM, Malaysian ringgits.
5 Blastocystis infection among Aboriginal children

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almost half of the households among both commu-
nities (the Aborigine and Malay) used water from
the streams, wells and rain with or without further
treatment by filtration or boiling. River water can be
highly exposed to contamination with infected feces
from wild/domestic animals and humans living
around the river (Azman et al. 2009; Ithoi et al.
2011). Therefore, drinking unboiled surface water
from streams is strongly believed to be the main risk
factor for Blastocystis infection, as shown in this
study.
Leelayoova et al. (2008) described the possibility
of waterborne transmission of Blastocystis by identi-
fying the same genotype of Blastocystis in fecal
specimens collected from schoolchildren and in the
drinking water provided in schools. A study con-
ducted in China, revealed a high infection rate
(32·6%) and found that the consumption of raw
water plants and drinking unboiled water were
predictors for Blastocystis infection (Li et al. 2007).
Moreover,inastudyamongpre-schoolchildrenfrom
Jordan, the prevalence rate of Blastocystis infection
was 25% and contaminated water was implicated as a
major source of infection (Nimri, 1993). In a world-
wide review on waterborne outbreaks of parasitic
protozoan infections, Karanis et al. (2007) documen-
ted 325 outbreaks since the 1950s and revealed that
the majority of outbreaks were reported in the USA
and Europe with Giardia and Cryptosporidium being
the predominant protozoa followed by Entamoeba,
Blastocystis, Cyclospora, Toxoplasma, Microsporidia
and free-living amoebas. Indicating a dramatic
increase in the number of waterborne outbreaks
caused by those protozoa, Baldursson and Karanis
(2011) showed that 199 outbreaks have been reported
between 2004 and 2010 with 40·2% and 30·6% of the
outbreaks occurring in New Zeeland and North
America, respectively.
Another significant finding that was associated
withtheriskofBlastocystisinfectionwasthelowlevel
of maternal education. Children of mothers with less
than 6 years of formal education were almost 3 times
more likely to be infected with Blastocystis. It is well
documentedthateducationofthefamily,particularly
the mothers, has an important role in preventing
intestinal parasitic infections among children (Okyay
et al. 2004). Daryani et al. (2006) reported that
prevalence of Blastocystis in children of working
mothers was less than those of non-working mothers.
They assumed that a higher educational level in
employed mothers played an important role in
hygienic education of their children. In contrast,
other studies found no association between the
mother’s educationallevel
Blastocystis infection (Leelayoova et al. 2008; Boeke
et al. 2010).
Subsequently, infection of Blastocystis, as either
single or multiple infections, was found to be
significantly associated
andthe rateof
withgastrointestinal
symptoms among these schoolchildren with abdomi-
nal pain (58·8%) and diarrhoea (50·0%) being the
common symptoms. To date, the pathogenic role of
Blastocystis is still controversial (Tan et al. 2010).
Numerous clinical and epidemiological studies con-
cluded that Blastocystis is a commensal organism and
probably is not responsible for clinical symptoms
when detected (Sun et al. 1989). On the other hand,
many studies have also shown the association of some
gastrointestinal symptoms with the infection, and
have suggested a positive correlation between the
parasite density and symptoms (Qadri et al. 1989;
Galantowicz et al. 1993; Nimri and Batchoun, 1994;
Kaya et al. 2007). In a large-scaled study aimed at
determining the prevalence and clinical importance
ofBlastocystisamong12136patientsinSaudiArabia,
17·5% of the patients were infected and almost half of
them were symptomatic with abdominal pain being
the most common symptom (87·9%) followed by
constipation (32·2%) and diarrhoea (23·4%) (Qadri
et al. 1989). In another study conducted among
hospitalized Turkish patients without any other co-
infecting pathogens, higher rates of symptoms were
present among
Blastocystis-infected
abdominal pain was present in 76·9%, diarrhoea in
50% and distention in 32·6%, indicating an associ-
ation between parasite density and pathology (Kaya
et al. 2007). Moreover, several studies found a higher
incidence of Blastocystis infection among immuno-
compromised individuals, including AIDS patients
and individuals with cancer (Horiki et al. 1999;
Tasova et al. 2000; Kurniawan et al. 2009). Besides
that, extraintestinal manifestations were reported as
invasive Blastocystis infections in 2 recent studies and
a debate over the issue that Blastocystis is an
opportunistic pathogen has began (Hu et al. 2008;
Janarthanan et al. 2011).
In conclusion, this study revealed that Blastocystis
infection exists among primary schoolchildren in
ruralPeninsularMalaysia.
maternal education were the main determinants of
the infection. Hence, improvement of sanitary
facilities and quality of drinking water, public
education and health promotion should be con-
sidered to prevent and control this infection.
children,
Watersupply and
ACKNOWLEDGEMENTS
The authors would like to thank all children and their
parents for their participation in this study. Thanks are due
to headmasters and teachers of the respective schools at
which specimens were collected, for their kind help and
cooperation.
FINANCIAL SUPPORT
This study was supported by a research grant from
University of Malaya (No. RG187/10HTM and PS230/
2010B), Kuala Lumpur, Malaysia.
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7Blastocystis infection among Aboriginal children